The isolation and purification of microbial, and in particular bacterial, nucleic acids from biological samples has become a routine tool in different fields of techniques. For example, the isolation of bacterial DNA in human blood samples is routinely performed during the screening of blood products for bacterial contaminations to prevent transfusion-transmitted diseases.
A common approach for the isolation of bacterial DNA from human blood samples involves the processing of the sample to release the DNA from whole cells or tissue extracts followed by DNA purification in spin columns or via magnetic beads. A number of DNA purification kits are commercially available and can be used for this purpose. However, these approaches have the drawback that the DNA from human blood cells is present in the sample in large amounts and might interfere with the detection of the microbial DNA. More sophisticated methods make use of immobilized proteins with ligands that bind to methylated human DNA to separate this DNA from the bacterial DNA that is present in the same sample (immuPREP Bacteria DNA Kit, Jena Analytik).
Methods for the consecutive lysis of blood cells and microbial cells are also known. EP 1 861 495 describes a method in which in a first step a chaotropic buffer is used to lyse human blood cells, but not microbial cells which are present in the same sample. The DNA released from the blood cells is degraded by the addition of a nuclease. Subsequently, the microbial cells are concentrated and the DNA is extracted and purified.
Although the above methods have provided a considerable contribution, there is still a need to develop new methods that allow for enriching and/or isolating nucleic acids from microbial cells, in particular from mixed samples which contain both microbial and higher eukaryotic cells or tissues, which are less laborious than those commonly known in the prior art. The method should preferably include fewer steps that require the manual intervention of the operator. Even more preferably, the method should be amenable to automation and require less time than currently used methods. The method of the present invention fulfills these needs and provides additional advantages as well.
It has unexpectedly been found by the inventors that microbial cells entrapped by filtration in a matrix can be efficiently lysed on this matrix, such as a commonly known silica or glass matrix which is part of a spin column, for DNA purification, without losing considerable amounts of DNA during cell lysis. Specifically, it was noted that, although the conditions during cell lysis are unfavorable for DNA binding, the DNA released from the cells was not washed out, but instead maintained on the filter matrix and could be reversibly bound to the matrix by the addition of monovalent and/or multivalent cations, e.g. magnesium or calcium cations, or cations from a chaotropic salt such as guanidine hydrochloride. Unbound material can be removed from the filter matrix by washing the matrix with a suitable washing buffer. Finally, the microbial DNA is eluted from the matrix in high purity. Without wishing to be bound by theory, it is assumed that the DNA is released from the microbial cells in a gentle way so that it maintains its highly complex molecular structure which enables it to remain loosely associated with the filter matrix even in the absence of high concentrations of monovalent or multivalent cations. This insight allows for an improved method that includes cell lysis directly on the filter matrix.
Compared to the common centrifugation-based methods, the method of the invention takes only half the time. Moreover, the method of the invention is amenable to automation, which requires only a vacuum station and an automated pipetting system, thereby making automation less expensive compared to centrifugation-based methods.